In high risk infants, wheezing-associated illness with rhinovirus (RV) is a significant risk factor for asthma development. Thus, RV infection in early life, in combination with other factors such as genetic background, al- lergen exposure and microbiome, may modulate the immune response, increasing the likelihood of asthma development. To test this, we developed an immature mouse model of RV1B infection. In contrast to mature animals, 6 day-old mice infected with RV1B develop sustained airways hyperresponsiveness, mucous meta- plasia and IL-13 production. This asthma-like phenotype is dependent a population of IL-13-producing type 2 innate lymphoid cells (ILC2s). In this proposal, we will test the general hypothesis that, in susceptible individual- als, early-life RV infection contributes to childhood asthma development via the expansion of IL-13-producing ILC2s. To test this general hypothesis, three specific aims are proposed: Aim 1. Determine the roles of IL-25, IL-33 and TSLP in RV-induced mucous metaplasia and airways hyperresponsiveness in neonatal BALB/c mice. We hypothesize that, in immature 6 day-old mice: i) RV infection increases airway cell production of IL-33 and TSLP; (ii) IL-25, IL-33 and TSLP are required for maxi- mal mucous metaplasia and AHR; (iii) a deficient IFN-? response allows RV-induced TSLP production; and iv) a permissive epigenetic state exists at the IL-25 promoter, allowing RV-induced transcription. Aim 2. Determine the contribution of ILC2s to RV-induced airway responses. We hypothesize that: i) in RV-infected immature mice, IL-25, IL-33 and TSLP function cooperatively to regulate expansion and IL-13 production by ILC2s; ii) ILC2s are required and sufficient for maximal RV-induced type 2 cytokine expression, mucous metaplasia and AHR; and iii) IFN-? attenuates ILC2 expansion and IL-13 production. Aim 3. Determine the effects of neonatal RV infection on responses to subsequent heterologous re- infection. We hypothesize that: i) early-life RV1B infection alters the immune response to subsequent RV2 or RSV infection, leading to type 2 rather than type 1 responses; ii) synergistic type 2 responses are driven by ILC2s; and iii) RV directly stimulates IL-13 production from ILC2s ex vivo. For Aims 1-3, to determine whether ILC2s constitute a common cellular response to early-life respiratory viral infection, we will compare RV1B, RV2 and RSV-A infections in 6 day-old immature mice and 8 month-old mature mice. Also, to support Aims 1 and 2, we will analyze IL-25, IL-33 and IL-33 levels and ILC2s in nasal and tracheal lavage samples taken from infants hospitalized with acute respiratory viral infections. Finally, to begin to understand why only some infants exposed to early-life viral infection may develop asthma, we will perform proof-of-concept experiments examining mucous metaplasia, airways hyperresponsiveness and ILC2s in RV-infected A/J, C57BL/6 and germ-free mice.